Laser-induced fluorescence spectroscopy (LIFS) has the ability to reveal both qualitative and quantitative information about the chemical or biochemical composition of an organic sample. LIFS has been applied in the diagnostic chemistry and medical fields to non-invasively provide information about biological systems in vivo. LIFS has advantages over some other optical techniques in that it can selectively and efficiently excite fluorophores in organic matter and greatly improve the fluorescence selectivity and detectability. Additional advantages of LIFS include wavelength tenability, narrow bandwidth excitation, directivity, and short pulse excitation. Early methods for LIFS detection and classification of biological samples were based on analysis of fluorescence intensity, spectral distribution, and polarization of light collected from the samples after excitation with a laser light source. In at least some instances, however, such detection methods may be unable to distinguish between fluorophores with similar emission spectra and may lack temporal resolution. Time-resolved LIFS (TR-LIFS) techniques build upon the characterization ability of earlier LIFS methods by adding the ability to analyze and characterize biological samples in real-time or near real-time. TR-LIFS takes advantage of short (on the order of nanoseconds) and ultra-short (on the order of picoseconds) pulsed laser technology and high speed electronics in order to allow the real-time evolution of a sample emission to be recorded directly.
Methods of TR-LIFS may involve monitoring the fluorescence lifetime or fluorescence decay of an excited biological sample in order to characterize the sample. Because the light emission process occurs very quickly after excitation by a light pulse (fluorescence decay is on the order of nanoseconds), a time-resolved measurement may provide information about molecular species and protein structures of the sample. While many molecules may have similar excitation and emission spectra, and may have similar fluorescence intensities, the decay profiles may be distinct or unique depending on the structure of the molecules. Thus, analysis of the fluorescence decay by TR-LIFS may distinguish between molecules which traditional LIFS fails to separate. TR-LIFS techniques may also be adapted to distinguish between “early” processes (typically the direct excitation of short-lived states or very rapid subsequent reactions) and “late” processes (typically from long-lived states, delay excitation by persisting electron populations, or by reactions which follow the original direct excitation) in a sample following excitation.
The fluorescence decay data may be complemented by spectral information (e.g. fluorescence intensity) for analysis of complex samples. A technique that has been used to record both fluorescence decay and fluorescence intensity data uses a scanning monochromator to select wavelengths from the broadband sample emission signal one wavelength at a time and direct the filtered signal to a photodetector for detection. However, in order to resolve another wavelength from the emission signal, the sample must be excited again in order to reemit the signal and the scanning monochromator must be re-tuned to a new wavelength. Such repeated measurements may take a significant amount of time, especially if a user wishes to resolve the sample emission signal into multiple spectral components, as switching between wavelengths can be a rate-limiting factor in producing real-time measurements. It would therefore be desirable to provide for characterizing a biological sample with time-resolved and wavelength-resolved analysis in (near) real-time.